Summary of work: Genotoxic/oxidative stresses contribute to the development of degenerative diseases, and are believed to be a major factor in normal aging as well. Cells respond to such stresses with changes in gene expression, but much remains to be learned about the signaling pathways mediating these effects and their functional consequences. This project encompasses studies related to these cellular responses. Specific investigations conducted over the past year include the following: (1) Mechanism of Akt Activation by Oxidative Stress - Akt is activated in response to a variety of oxidative insults including hydrogen peroxide, peroxynitrite and singlet oxygen. Our studies indicate that this is mediated largely through usurping of EGF and PDGF growth factor signaling pathways. (2) Mechanism of Cisplatin-induced Apoptosis - We have shown that cisplatin their apoptosis. Preventing ERK activation inhibits cisplatin cytotoxicity, which may have important therapeutic implications. (3) Role of c-Jun-N-terminal kinase 2 (JNK2) in Regulating Tumor Cell Growth - Using an antisense (AS) strategy to block JNK2 expression and activity, we have observed that inhibition of JNK2 leads to growth inhibition and apoptosis of certain cancer cells that lack p53 function and are unable to express the p53 effector protein p21/Waf1/Cip1. Current studies are addressing the mechanisms involved using both SAGE and cDNA array approaches to identify genes expressed differentially in control and JNK2AS-treated cells. (4) Role of c-Jun in Influencing Cell Survival Following Stress - We are using cell lines that either lack c-Jun expression or harbor a mutant c-Jun form (which can not be phosphorylated and activated) to test the role of this transcription factor in influencing cell survival following different stresses. We have found that c-Jun deficiency sensitizes cells to DNA damaging stresses but not to other non-DNA damaging inducers of apoptosis. Current studies are exploring the basis for this differential sensitivity. (5) Role of PLCgamma1 in Oxidative Stress - We have found that cells deficient in PLCgamma1 expression show heightened sensivity to a number of oxidant insults and are attempting to understand the basis for this effect. (6) Mechanisms Contributing to Age-related Declines in Stress Signaling Pathways - Using primary hepatocytes derived from different age rats we have found that the activities of several of signaling pathways (heat shock response, ERK MAPK activation and p70 S6Kinase) are attenuated in aged cells. This study focuses on the biochemical and molecular bases for this decline as well as the functional consequences.